The Cell Model's Estabishment Of Parkinson's Disease And The Toxic Damage Of Rotenone To Dopaminergic Neurons

Objective:Parkinson's disease(PD) is a common progress neurodegenerative disease in the aged. The main pathological hallmark of this disorder is a pronounced loss of dopaminergic neurons(DA) in the substantianigra,and Lewy body appears in the remaining neurons. The clinical features include static tremor,bradykinesia,myotonia,posture and gait irregularity et. The cause of the most idiopathic PD is not clear. The environmental factors are the focus. N-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP) is the first exogenous chemical materials proved to be toxicalto the DA neurons of central nervous system. But it does not exist innature and is seldom touched in daily life. Pesticide rotenone derived from the roots of derris,can induce PD pathological and behavior changesin rats. Study on rotenone shows it may be concerned with PD.In 1997 when α-synuclein gene mutation was first found in a familial autosomal dominant inheritance Parkinson disease,people began relating α-synuclein with PD. Both familial PD patients and idiopathic PDpatients have Lewy bodies in brain and LB's major component is α-synuclein. Many investigations show α-synuclein corrolates closely with PD.Cell death at least goes through two different kinds of forms,necrosis and apoptosis. After the PD patient dies, autopsy results show dopaminergic neurons(DA) in the substantia nigra proceed nucleus shrinkage,chromatin concentration and form apoptosis body. The discovery proves this kind of neuron death belongs to apoptosis not necrosis.There are lots of studies on the pathogenesis of Parkinson's disease,and people now pay more attention to interreaction between genetic andenvironmental factors which lead to PD. The mechanism through whichenvironmental factors cause the accumulation of a-synuclein,induce cell apoptosis and finally cause DA damages is not very clear at present.Our study takes pesticide rotenone as an environmental factor, rat adrenal pheochromocytoma cells (PC12) as experimental cells to establishcell model of PD. Though detecting inclunsions in cytoplasm and cell apoptosis,we expect to approach rotenone toxic mechanism to DA neurons,providing clues for the possible pathogenesis of PD and establishing foundation for proteome study of Lewy bodies and associated proteins.Method:Using 100ng·ml-1 nerve growth factor,PC12 cells weredifferentiated into dopaminergic neurons. After that we added different concentrations of rotenone to the solution for 24 hours,48 hours and 72 hours, cell morphology changes were observed and cell viability was assessed with MTT. Using the concentrations which cells viability drops apparently,we cultured cells for 72 hours,then immunohistochemistry and immunofluorescence were used to observe the accumulation of α-synuclein in cytoplasm. AO/EB double staining was also adopted to test cell apoptosis at different time.Result:Before NGF's differentiation,PC12 cells shaped regularly, round or oval-shap, whose ecptomas were little and short and with a fewcell conjunctions. After differentiation, cells shaped irregularly, assumingpolygon,long spindle-shaped et,whose ecptomas were big and long,with multiple cell conjunctions. After dealing with rotenone,the ecptomasvanished gradually and cell bodies became smaller and smoother. Cell viability declined in a time and concentration dependent manner. At the concentration of 50nmol·L-1when treated with rotenone for 24 hours,cellviability dropped significantly (P <0.05). At the concentrations of 50nmol·L-1 and 100nmol·L-1 cell immunohistochemistry demonstrated a brown round α-synuclein-immunopositive mass in cytoplasm;At the sametime cell immunofluorescence also showed in cytoplasm a strong greenprotein aggregation which was α-synuclein immunolabelled. As cell concentrations grew and time was prolonged,viable apoptotic cell,non-viable apoptotic cell and necrosis cell can be seen gradually at different time.Conclusion: Using rotenone we can successfully establish PC12-damaged cell model of PD. Rotenone can cause toxic damage to DA neurons,making α-synuclein protein aggregation in cytoplasm and inducing cell apoptosis,which suggests that rotenone might act through the metabolism of α-synuclein and the induction of cell apoptosis in the pathogenisis of Parkinson's disease.